JP2018079145A - Sol for tissue hole closing, ulcer protection, and vascular embolization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sol for biological injection that can be used for tissue hole closing, ulcer protection, and vascular embolization and is suitable for delivery through a catheter.SOLUTION: The present invention provides a sol for tissue hole closing, ulcer protection, and vascular embolization that contains 0.6 mass%-3 mass% of collagen, water, 200 mM-330 mM of sodium chloride, and a buffer agent and has a pH of 6.0-9.0.SELECTED DRAWING: None

Description

  The present invention relates to a sol for bioinjection used for endoscopic treatment or the like, and more specifically, an ulcer that can occur during endoscopic treatment or the like in order to close a hole generated during endoscopic treatment or the like. The present invention relates to a sol for closing a biological tissue hole, protecting an ulcer, and vascular embolization for use in vascular embolization for protecting cancer or for gastrointestinal bleeding.

  Minimally invasive treatments such as endoscopic treatment and IVR (interventional radiology) treatment (X-ray, CT, etc., which are performed by inserting a catheter into the body while using an image diagnostic apparatus) are rapidly spreading. It's getting on. These minimally invasive treatments may require the use of a bioinjection gel to close through-holes, protect ulcers, and embolize blood vessels created in living tissue. Delivery of chemicals and materials via a catheter is essential for minimally invasive treatment, but it is not easy to deliver a solid material via a catheter.

  Preparations that have gelling ability used in clinical practice are called bioadhesives (also called biosealants and tissue adhesives), preparations using a reaction between a crosslinking agent and a polymer, and preparations using a polymerization reaction of monomers. (Patent Document 1, etc.). Although it has a function of gelling in a short time and binding to a living tissue, gelation starts immediately after the preparation is prepared, so that it is not suitable for an application for delivery through a long capillary such as a catheter. Therefore, it is difficult to use for biological tissue hole closure, ulcer protection, and vascular embolization which are often performed endoscopically.

  In addition, bioinjection gels that cause sol-gel transition by self-assembly of polymers (hydrophobic interaction, electrostatic interaction, etc.) without using a crosslinking agent have been reported (Patent Documents 2 to 4, etc.) ). Since it gels in response to body temperature, there are advantages such as easy delivery via a catheter, but it is weak and unstable, poorly fixed to living tissue, closed tissue hole, ulcer protection and Difficult to use for vascular embolization.

  Furthermore, as a preparation widely used as a biological sealant or hemostatic agent, there is a fibrin glue using a cross-linking reaction between fibrinogen and thrombin, which is one of biological reactions. Since the gelation time after mixing the chemicals is long, it can be delivered via a catheter and has a hemostatic effect. However, since gelation has no body temperature responsiveness, it is difficult to form a gel locally with good reproducibility. Even if a gel can be formed locally, the strength of the gel is weak, so a stable ulcer coating cannot be formed unless used in combination with a protective sheet such as a polyglycolic acid sheet (Non-patent Document 1). Adhesive strength to living tissue is also extremely low (Patent Document 5) and lacks stability as a closure material, embolization / occlusion, and protective material. Furthermore, since it is a kind of blood product, it has a high risk in terms of safety, and infections such as hepatitis C have been reported.

  On the other hand, the present inventor has already found that a specific collagen / genipin mixed aqueous solution has a gelling property in which collagen is fibrillated at a temperature close to body temperature and then genipin crosslinking is introduced (Patent Document 6). It has also been found that the gelation rate of a specific collagen aqueous solution can be accelerated by adjusting the inorganic salt concentration (Patent Document 7 and Non-Patent Document 2). However, the effectiveness of these aqueous solutions for specific medical uses has not been known.

Japanese Patent No. 4585743 Japanese Patent No. 5019851 Special table 2008-505919 JP2014-221830 JP2007-325824 JP2014-103985A JP2016-077741A

Tsuji et al. Gastrointestinal Endoscopy Volume 79, Issue 1, Pages 151-155, 2014 Yunoki et al. Journal of Biomedical Materials Research Part A Volume 103, Issue 9, pages 3054-3065, 2015

  As described above, there has been no bioinjection gel suitable for delivery by a catheter that can be used for biological tissue hole closure, ulcer protection, or vascular embolization. In addition, there has been no case where a sol-like substance is delivered via a catheter and the sol-gel transition gel has successfully closed a biological tissue hole, protected an ulcer, or embolized a blood vessel.

  In view of such a background, an object of the present invention is to provide a sol for injecting a living body suitable for delivery by a catheter, which can be used for closing a living tissue hole, protecting an ulcer, or performing vascular embolization.

  In response to the above problems, the present inventors have developed a sol containing a specific concentration of collagen, water, a specific concentration of sodium chloride and a buffer and having a specific pH to close through holes and physically protect ulcers. And three characteristics suitable for vascular embolization: (1) long fluidity retention time that can be delivered from outside the living body through the catheter, and (2) sharp body temperature responsiveness that gels immediately after delivery, And (3) The present invention has been completed by finding that it has the property of hardening after gelation and fixing to a living tissue, and can close a living tissue hole, protect an ulcer or embolize a blood vessel.

That is, the present invention relates to the following.
[1]
A biological tissue pore closing sol containing 0.6 mass% to 3 mass% collagen, water, 200 mM to 330 mM sodium chloride and a buffer, and having a pH of 6.0 to 9.0.
[2]
An ulcer protective sol containing 0.6% to 3% by mass of collagen, water, 200 mM to 330 mM sodium chloride and a buffer, and having a pH of 6.0 to 9.0.
[3]
A vascular embolization sol containing 0.6 mass% to 3 mass% collagen, water, 200 mM to 330 mM sodium chloride and a buffer, and having a pH of 6.0 to 9.0.
[4]
The sol according to any one of [1] to [3], wherein the buffering agent includes a phosphate.
[5]
The sol according to any one of [1] to [4], which contains genipin or a genipin derivative in a range of 40 mg / L to 1400 mg / L.
[6]
The sol according to any one of [1] to [5], wherein the collagen contains telopeptide-removed collagen.
[7]
The sol according to any one of [1] to [6], which contains 1.4% by mass to 1.7% by mass of collagen and is locally administered to a living tissue through a catheter.
[8]
The sol according to any one of [1] to [7], which gels and adheres to a living tissue when it comes into contact with the living tissue.

The present invention also relates to the following.
[9] A kit for closing a biological tissue hole, protecting an ulcer, or embolizing a blood vessel with a sol that gels upon contact with the biological tissue and adheres to the biological tissue,
A kit comprising collagen, sodium chloride, a buffer and genipin for forming the sol.
[10]
A kit for closing a biological tissue hole, protecting an ulcer or embolizing a blood vessel with a sol that gels upon contact with the biological tissue and adheres to the biological tissue,
A kit containing 0.6% to 3% by weight of collagen, water, 200 mM to 330 mM sodium chloride and a buffer and having a pH of 6.0 to 9.0, and genipin.

  According to the present invention, a sol that can be delivered through a catheter can close a through-hole in biological tissue, protect an ulcer, or embolize a blood vessel.

The conditions of porcine stomach perforation formation (FIG. 1a), collagen sol delivery (FIG. 1b), gelation (FIG. 1c), and perforation closure (FIG. 1d) are shown in the porcine stomach perforation closing experiment of Test Example 1. The state of the leak test of the removed stomach in the perforation closure experiment of the porcine stomach of Test Example 1 is shown. 7 shows an HE-stained image of porcine stomach tissue including a perforated part in a porcine stomach perforation closing experiment of Test Example 1. Fig. 4 shows porcine colon perforation formation (Fig. 4a), collagen sol delivery (Fig. 4b), gelation (Fig. 4c), and perforation closure (Fig. 4d) in the experiment of porcine colon perforation closure in Test Example 2. The state of the leak test of the excised colon in the experiment for porcine colon perforation closure in Test Example 2 is shown. In the porcine stomach perforation closing experiment of Test Example 3, porcine stomach perforation formation (Fig. 6a), collagen sol delivery (Fig. 6b), gelation (Fig. 6c), perforation closure (Fig. 6d), large perforation closure (Fig. 6e) and the state in which the gel was not destroyed even when physiological saline was sprayed onto the gel (FIG. 6f). The result of the ex vivo ulcer protection test of the porcine stomach of Test Example 4 is shown. The result of the dynamic viscoelasticity test of the collagen sol of Test Example 5 is shown. The result of evaluating the body temperature responsiveness of gelation is shown in FIG. 8a, and the result of tracking G 'up to 30 minutes after sol preparation is shown in FIG. 8b. The result of the dynamic viscoelasticity test of the collagen sol of Test Example 6 is shown. The gelation behavior of sols with different NPB concentrations is shown in FIG. 9a, and the result of plotting the gelation time after reaching 37 ° C. against the NPB concentration is shown in FIG. 9b. The gel penetration test result of Test Example 7 is shown. The stress-strain curves for gels with different genipin concentrations are shown in FIG. 10a and the elastic modulus is shown in FIG. 10b. The result of a viscosity measurement of Test Example 8 is shown. The result of the perforation closure experiment of the porcine stomach using the collagen sol of the comparative example 1 of the test example 9 is shown.

  Hereinafter, modes for carrying out the present invention (hereinafter simply referred to as “this embodiment”) will be described in detail. The following embodiment is an exemplification for explaining the present invention, and is not intended to limit the present invention only to this embodiment.

  The sol (sol composition, pharmaceutical composition) of the present embodiment contains 0.6 mass% to 3 mass% collagen, water, 200 mM to 330 mM sodium chloride and a buffer, and has a pH of 6.0 to 9 .0.

  Collagen contained in the sol of this embodiment is not particularly limited, but is preferably telopeptide-removed collagen that hardly progresses to fibrosis around room temperature, and more preferably substantially consists of telopeptide-removed collagen. . Telopeptide-removed collagen is a telopeptide that collagen molecules have at both ends enzymatically decomposed and removed by proteolytic enzymes. For example, telopeptide that collagen molecules have at both ends is decomposed and removed by pepsin digestion. It is a thing. Among telopeptide-removable collagens, mammalian-derived telopeptide-removable collagen that is approved as a raw material for medical devices is preferable, and telopeptide-removable collagen derived from pig skin that has already been clinically applied and has excellent thermal stability. More preferably used. Telopeptide-removed collagen is commercially available as another name for atelocollagen and can be easily obtained.

  Collagen is not particularly limited as long as it has collagen-forming ability (fibrogenic collagen). Among fibrogenic collagens, type I which is collagen constituting bone, skin, tendon and ligament, type II which is collagen constituting cartilage, type III contained in living tissue composed of type I collagen, etc. It is preferably used from the viewpoints of availability, abundant research results, and similarity to the biological tissue to which the produced gel is applied. Collagen may be obtained by extraction and purification from a living tissue by a conventional method, or a commercially available product may be obtained. Collagen may be purified from each type or a mixture of multiple types.

  The denaturation temperature of collagen is preferably 32 ° C. or higher, more preferably 35 ° C. or higher, and further preferably 37 ° C. or higher. When the denaturation temperature is 32 ° C. or higher, the fluidity of the sol at room temperature can be maintained for a longer time, and collagen denaturation is less likely to occur in vivo. The upper limit of the denaturation temperature of collagen is not particularly limited, but is preferably 50 ° C. or lower, more preferably 45 ° C. or lower, and further preferably 41 ° C. When the denaturation temperature is equal to or lower than the above upper limit value, gelation at the time of contact with a living tissue can be advanced more rapidly. The denaturation temperature of collagen is measured by a conventional method, that is, a change in circular dichroism, optical rotation, or viscosity accompanying an increase in temperature of the collagen aqueous solution. The denaturation temperature of collagen may be adjusted by selecting collagen having a denaturation temperature within the above numerical range.

The sol of this embodiment is a sol containing a collagen aqueous solution containing collagen and water, and a sol having a high collagen concentration is desirable from the viewpoint of sol retention for causing gelation locally at the site of administration. If the collagen concentration is too low, the viscosity of the sol decreases, and the sol may dissipate from the introduction site before gelation. In addition, since the hardness of the gel after gelation is improved when the collagen concentration of the sol is high, a sol having a high collagen concentration is desirable from the viewpoint of reliably performing tissue through-holes, vascular embolization, and the like.
On the other hand, from the viewpoint of administering the sol of this embodiment via a catheter, a sol having a low collagen concentration is desirable. Depending on the diameter and length of the catheter, as the collagen concentration increases, the sol viscosity increases, the extrusion resistance increases, and administration may be difficult.
From the above viewpoint, in the sol of the present embodiment, the collagen concentration is 0.6% by mass to 3.0% by mass and 0.8% by mass to 2.2% by mass based on the total amount of the sol. And preferably 1.4% by mass to 1.7% by mass, and particularly preferably 1.4% by mass to 1.6% by mass.

  The sol of this embodiment contains sodium chloride, which is an inorganic salt, at a predetermined concentration, whereby collagen fibrillation is accelerated when it comes into contact with a living tissue, and gels quickly in response to body temperature.

  The concentration of sodium chloride contained in the sol can be appropriately adjusted in the range of 200 mM to 330 mM higher than the physiological salt concentration (140 mM), preferably 220 mM to 310 mM, and can be, for example, around 280 mM. When the sodium chloride concentration is less than the physiological salt concentration, collagen fibrosis in response to body temperature may take a long time. On the other hand, if the sodium chloride concentration exceeds 330 mM, the sol may easily lose its fluidity in the catheter. By setting the concentration of sodium chloride within such a range, it becomes possible to rapidly gel in response to body temperature while maintaining the fluidity of the sol in the catheter.

  The pH of the sol of the present embodiment (pH at 23 ° C., the same throughout the present specification unless otherwise specified) is 6.0 to 9.0, and more preferably 6.5 to 8.0. It is known that collagen fibrosis occurs actively near neutrality. By setting the pH within a predetermined range, collagen fibrosis can be further promoted. The pH can be adjusted by a conventional method, for example, control of the concentration of inorganic salt contained in the sol, preferably sodium chloride and sodium hydrogenphosphate, strong acid such as hydrochloric acid and sodium hydroxide, and / or The pH can be adjusted by adding a strong alkali. The pH can be measured with a known pH meter (for example, trade name “NAVIh F-71” manufactured by HORIBA).

Moreover, the sol of this embodiment contains a buffer for the purpose of maintaining pH. The buffer is not particularly limited as long as the sol has desired characteristics. For example, phosphate, acetate, borate, HEPES, Tris, and the like can be used. Examples of phosphates include sodium phosphate, sodium hydrogen phosphate (generic name for sodium dihydrogen phosphate and disodium hydrogen phosphate), and potassium hydrogen phosphate (generic name for potassium dihydrogen phosphate and dipotassium hydrogen phosphate). Etc. can be used. Sodium acetate or the like can be used as the acetate, and sodium borate or the like can be used as the borate, which can be used in combination with pH adjustment with sodium hydroxide or the like. Moreover, you may use buffer solutions, such as sodium chloride containing phosphate buffer (NPB) which combined said sodium chloride and the buffering agent.
Of these buffers, phosphate and NPB containing the same are particularly preferably used. Phosphate has excellent buffering ability at pH 6-9 where collagen fibrillation is actively generated, and has an advantage that safety to living bodies has been confirmed so as to be included in cell washing solutions such as phosphate buffered saline. There is.

The concentration of the buffer is not particularly limited as long as the pH is maintained in a desired range and the sol has desired characteristics.
From the viewpoint of sufficiently exhibiting the pH buffering effect, the buffering agent concentration can be 5 mM or more. On the other hand, if the buffer concentration is high, salt in the buffer solution may precipitate before dispensing, or the ionic strength may become too high and cause tissue damage when using the sol. It can be. The buffer concentration is preferably more than 10 mM and less than 120 mM, for example, 20 mM to 110 mM, and more preferably 30 mM to 100 mM. By setting the buffer concentration in such a range, it becomes easy to maintain the pH of the sol within the range of 6.0 to 9.0, and the catheter can be operated while maintaining the fluidity of the sol within the catheter. It is possible to suppress salt precipitation and tissue damage while exhibiting the effect of the sol of the present embodiment that gels at a delivery place promptly in response to body temperature after delivery via.

When the sol comes into contact with a living tissue, it gels in response to body temperature. From the viewpoint of increasing the strength of the gel and improving the adhesion to living tissue, the sol may contain a crosslinking agent. The crosslinking agent is not particularly limited, and one kind can be used alone or two or more kinds can be used in combination. However, the plant-derived genipin, which is considered to have low cytotoxicity of the crosslinking agent itself, or a crosslinking agent between collagen molecules. 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (hereinafter referred to as “EDC”) which is removed by washing with water and N-hydroxysuccinimide (NHS) which is a crosslinking aid thereof Preferably used. Genipin is an aglycone of geniposide, and can be obtained, for example, by oxidation, reduction and hydrolysis of geniposide or by enzymatic hydrolysis of geniposide. Geniposide is an iridoid glycoside contained in Rubiaceae gardenia and is obtained by extraction from gardenia. Genipin is represented by a molecular formula of C ₁₁ H ₁₄ O ₅ , and may be synthesized by a conventional method or a commercially available product may be obtained. Further, genipin may be derivatized to the extent that the crosslinking effect is ensured to the extent that the desired properties of the sol of the present embodiment are not impaired. As a derivative of genipin, for example, the one described in JP-T-2006-500975 can be used. In the present specification, genipin includes a polymer of genipin. Genipin is known to polymerize under various conditions, and the polymerization conditions and method are not particularly limited. For example, a method of polymerizing by aldol condensation under strong alkaline conditions (Mi et al. Characterization of ring-opening polymerization) Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 43, 1985-2000 (2005)) of genipin and pH-dependent cross-linking reactions between chitosan and genipin.
EDC is a kind of water-soluble carbodiimide, and any water-soluble carbodiimide can be used as a cross-linking agent regardless of its type. Among them, EDC is particularly preferably used because it is inexpensive and highly safe. A water-soluble carbodiimide is used individually by 1 type or in combination of 2 or more types. EDC may be used alone or in combination with NHS. It is known that the crosslinking reaction by EDC is promoted by mixing NHS.

  When the sol of this embodiment contains genipin, the genipin concentration can be 1800 mg / L or less from the viewpoint of maintaining the fluidity of the sol until delivery to the affected area, and is preferably 40 mg / L to 1400 mg / L, for example, 100 mg / L to 1000 mg / L. By setting the genipin concentration within such a range, the gel strength can be enhanced by crosslinking while maintaining the fluidity of the sol in the catheter.

  The sol of this embodiment may further contain various solvents and additives used in conventional collagen aqueous solutions. Examples of such solvents and additives include acids such as dilute hydrochloric acid, citric acid, and acetic acid.

  The said additive and solvent are used individually by 1 type or in combination of 2 or more types. Further, the content ratio of the additive and the solvent in the sol is not particularly limited as long as the desired characteristics of the sol of the present embodiment are not impaired.

  The sol of this embodiment is useful in minimally invasive treatment using catheters such as endoscopic treatment and IVR treatment, and is particularly useful for closing through-holes generated in living tissues, protecting ulcers, and embolizing blood vessels. is there.

  For example, in endoscopic treatments such as EMR (endoscopic mucosal resection) and ESD (endoscopic submucosal dissection), the occurrence of perforation as an accident has been a problem. Closing of the perforation is performed by clipping, but it takes time to deliver several clips one by one through the scope, and advanced operation techniques of the endoscopic device are required to reliably close the perforation. . Post bleeding and delayed perforation due to poor ulcer healing after endoscopic treatment have been reported.

  In addition, vascular embolization is widely used in IVR treatment. Vascular embolization is a technique in which an embolic material is sent into a blood vessel through a catheter inserted into an artery for the purpose of treating cancer such as the liver and stopping hemorrhage from the digestive tract or lungs. Specifically, the embolizing material is delivered to the vicinity of a lesion in an organ, and is delivered to a blood vessel supplying nutrients to a cancer lesion and a blood vessel causing bleeding, thereby blocking the blood flow. Gelatin sponges conventionally used for embolization materials need to be cut according to the size of the affected area, and there is a problem that embolization cannot be achieved if the size is incompatible. Coils are also used for embolization materials, but they are expensive and once delivered will remain in the body forever.

  The sol of this embodiment has a long fluidity retention time that can be administered to a living tissue through a catheter, a body temperature responsiveness that quickly gels at a living body temperature after delivery, and a property that adheres to living tissue after gelation. Particularly useful in the above-mentioned applications, and more specifically, protection of ulcers formed during endoscopic treatments such as ESD and EMR (including ulcer repair associated therewith), clipping It is useful for closing a perforated part including temporary closure for easy operation, vascular embolization during IVR treatment, and the like.

  In the conventional bioadhesive, the viscosity immediately increases after compounding and gelation occurs in about 1 minute. However, the sol of this embodiment has a long fluidity retention time that can be administered to a living tissue through a catheter. (For example, 10 minutes at room temperature) and can be delivered via, for example, an endoscope or a fluoroscopic image via a catheter having an inner diameter of 2.2 mm and a total length of 2.8 m. In the present embodiment, administration through a catheter includes administration using a nebulizing catheter. The inner diameter and length of the catheter used for administration can be appropriately changed according to the administration site, the viscosity of the sol, etc. For example, a catheter having an inner diameter of 0.5 mm to 2.8 mm and a length of 1 m to 3 m is used. it can. The sol of the present embodiment has a characteristic that it can be administered to a living tissue even when a catheter having a small inner diameter (for example, an inner diameter of 0.5 mm to 2.5 mm) or a long catheter (for example, a length of 1.5 m to 3 m) is used. Have

The viscosity of the sol of this embodiment can be adjusted as appropriate according to the pore diameter of the catheter. For example, the viscosity measured at 23 ° C. at a shear rate of 1 s ⁻¹ is 0.2 Pa · s to 52 Pa · s, preferably 2 Pa · s to 20 Pa · s, more preferably 5 Pa · s to 12 Pa · s. When the viscosity is in the above range, it has fluidity during administration, and can remain in the affected area after administration to form a gel at a desired position. The viscosity is measured using a known rheometer capable of controlling the shear rate using a cone-plate system described in Japanese Industrial Standard (JIS) K7117-2, as exemplified in the examples below. it can. The polymer solution such as the collagen sol of the present embodiment is a non-Newtonian fluid, and when the shear rate during measurement increases, the viscosity decreases and converges to a certain viscosity value. Therefore, the viscosity of the sol of the present embodiment is described as a viscosity measured specifically at a shear rate of 1 s ⁻¹ at a low shear rate where the difference in viscosity is clearly observed.

  After administration to the living tissue through the catheter as described above, the sol of this embodiment starts gelation rapidly at the living body temperature (body temperature) to form a gel locally, thereby closing the living tissue hole, protecting the ulcer, and vascular embolism It can be performed. Gelation usually occurs within 5 minutes after reaching 37 ° C. after administration. The strength of the gel to be formed is not particularly limited, but for example, the elastic modulus determined by a compression test or a penetration test described in Examples described later is preferably in the range of 10 kPa to 200 kPa, and preferably in the range of 20 kPa to 100 kPa. is there. If the elastic modulus is too small, the gel may be easily deformed and the biological tissue hole may be closed or the vascular embolus may be broken. When the elastic modulus significantly exceeds the living tissue around the gel, when the living tissue is distorted, the adhesion between the gel and the living tissue may be broken due to the difference in elastic modulus.

As described above, when a gel is locally formed, collagen is first fibrillated on a living tissue (a kind of self-organization) to form a gel (primary gelation). When the sol contains a cross-linking agent such as genipin, cross-linking is introduced into the collagen fiber gel (secondary gelation), the gel strength increases, and the collagen and the living tissue are chemically strengthened. Glue.
The sol of the present embodiment can penetrate and settle in the submucosal layer of a living tissue.
Due to the above characteristics, the sol of this embodiment can form a gel having a tissue fixing property and strength enough to close a perforation and embolize a blood vessel locally with good reproducibility in a living body where flow occurs. it can. The formed gel is a gel having a component composition excellent in safety and biocompatibility, and gradually undergoes actions such as hydrolysis and enzymatic degradation as in the case of normal collagen.

  The sol of this embodiment may further contain a drug according to the state of the affected area to be administered. Such a drug is not particularly limited as long as it can be contained in a conventional injectable gel, and examples thereof include physiologically active peptides, proteins, other antibiotics, antitumor agents, and hormone agents. Can be mentioned. A medicine is used individually by 1 type or in combination of 2 or more types. Moreover, the content rate of a chemical | medical agent will not be specifically limited if it is the range which does not inhibit the desired characteristic of the gel of this embodiment, exhibiting the effect of the chemical | medical agent.

The present embodiment also relates to a kit for closing a biological tissue hole, protecting an ulcer, or embolizing a blood vessel with a sol that gels upon contact with the biological tissue and adheres to the biological tissue.
In one embodiment, the kit includes collagen, sodium chloride, and a buffer for forming the sol, and optionally further includes genipin, a catheter for sol administration, and the like. Each component contained in the kit may be in the form of a solution, or the components contained in the kit in a dry state may be appropriately dissolved before use to form a sol.
In one embodiment, the kit contains 0.6% to 3% by weight of collagen, water, 200 mM to 330 mM sodium chloride and a buffer, and has a pH of 6.0 to 9.0. It may be a kit containing sol for use and genipin.

  Hereinafter, although this embodiment is described more concretely based on an example and a comparative example, the present invention is not limited to the following examples and comparative examples.

[Preparation of collagen solution]
A collagen solution made of pig skin having a concentration of 1.0% by mass (telopeptide-removable collagen, Nippon Ham Co., Ltd., collagen denaturation temperature: 40 ° C.) was prepared as a collagen stock solution. The collagen solution was concentrated using an evaporator (water temperature: 29 ° C.) to obtain a collagen solution having a concentration of 2.4% by mass. This was divided into 15 mL centrifuge tubes or 50 mL centrifuge tubes and stored in a refrigerator.

[Preparation of genipin aqueous solution]
Genipin (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in pure water to prepare a genipin aqueous solution having a concentration of 24 mM (5430 mg / L). This was diluted with pure water to prepare genipin aqueous solutions having different concentrations.

[Preparation of NPB]
A 50 mM concentration disodium hydrogen phosphate aqueous solution (containing 140 mM sodium chloride) and a 50 mM sodium dihydrogen phosphate aqueous solution (containing 140 mM sodium chloride) were prepared using pure water as a solvent. These were stirred and mixed while measuring with a pH meter (trade name “NAVIh F-71” manufactured by HORIBA) to prepare a 140 mM sodium chloride-containing 50 mM phosphate buffer solution having a pH of 7.0. Defined. In addition, throughout the examples, unless otherwise specified, pH was measured at 23 ° C. using the pH meter. By the same operation, 12 × NPB (0.6M phosphate buffer containing 1.68 M sodium chloride at pH 7.0) was prepared and diluted with pure water to prepare different multiples of NPB (n × NPB).

[Example 1]
[A. Preparation of collagen sol: when used for other than animal experiments]
The collagen solution 6g which prepared in the above and entered into the 15 mL centrifuge tube was left still in the styrofoam container filled with crushed ice. A magnetic stirrer (10.8 g, inner diameter 10 mm × 39 mm) for promoting stirring was accommodated in the tube. Next, 2 mL of a genipin aqueous solution left in a refrigerator at 4 ° C. and 2 mL of 10 × NPB left at room temperature were sucked up with a micropipette, added to a centrifuge tube containing a collagen solution, and the centrifuge tube was vigorously shaken and stirred. . The centrifuge tube after stirring for about 30 seconds is set at a predetermined position of the centrifuge, centrifuged at 3200 rpm for 1.5 minutes, air bubbles are collected on the liquid surface, and 1.44% collagen sol ( Solvent, 2 × NPB; genipin concentration, 4 mM (905 mg / L)).
[B. Preparation of collagen sol: for animal experiments]
12 g of the collagen solution prepared in the above manner and contained in a 50 mL centrifuge tube was allowed to stand in a styrofoam container filled with crushed ice. A magnetic stirrer (10.8 g, inner diameter 10 mm × 39 mm) for promoting stirring was accommodated in the tube. Next, 4 mL of a 20 mM genipin aqueous solution that was allowed to stand in a container filled with crushed ice and 4 mL of 10 × NPB that was allowed to stand at room temperature were sucked up with a micropipette and added to the centrifuge tube containing the collagen solution. The mixture was shaken and stirred to obtain 1.44% collagen sol (solvent, 2 × NPB; genipin concentration, 4 mM (905 mg / L)). This was used for the porcine gastric perforation closing experiment of Example 1.

[Example 2]
A collagen sol having the same composition as in Example 1 was prepared except that the collagen concentration was increased from 1.44% to 1.6%.
Example 3
A collagen sol having the same composition as in Example 1 was prepared except that the NPB concentration was lowered from 2 × NPB to 1.8 × NPB.
Example 4
A collagen sol having the same composition as in Example 1 was prepared except that the NPB concentration was decreased from 2 × NPB to 1.6 × NPB.
Example 5
A collagen sol having the same composition as in Example 1 was prepared except that the genipin concentration was lowered from 4 mM to 2 mM (452 mg / L).
Example 6
A collagen sol having the same composition as in Example 1 was prepared except that genipin was not contained.

[Comparative Example 1]
A collagen sol in which the NPB concentration of the collagen sol of Example 1 was increased from 2 × NPB to 2.4 × NPB and the genipin concentration was increased from 4 mM to 8 mM (1810 mg / L) was prepared.
[Comparative Example 2]
A collagen sol having the same composition as in Example 1 was prepared except that the NPB concentration was decreased from 2 × NPB to 1 × NPB.
[Comparative Example 3]
A collagen sol having the same composition as in Example 1 was prepared except that the NPB concentration was lowered from 2 × NPB to 1 × NPB and the collagen concentration was lowered from 1.44% to 0.5%.

〔Test method〕
[Pig stomach closed experiment]
An acute perforation closure experiment was conducted using SPF pigs (30 kg body weight). Under general anesthesia, saline was locally injected into the porcine stomach submucosally, and ESD was performed on the bulging virtual lesion using an ESD-dedicated knife. A perforation having an inner diameter of 5 mm was made in the formed ulcer bottom. After confirming the collapse of the stomach due to deaeration, immediately collagen sol was passed through the catheter (total length: 2400 mm; product name: Finejet S2825 (top); inner diameter: 2.2 mm; tip 100 mm cut with scissors). Delivered. After confirming that the stomach was dilated by insufflation, it was rested for 1 hour, and after euthanizing the pig, the stomach was removed. A leak test was performed in which the stomach pylorus was closed with forceps and water was injected from the cardia to fill the stomach with water. After the leak test, the part including the perforated part was excised and fixed and subjected to tissue observation by hematoxylin-eosin (HE) staining.

[Pig colon closure experiment]
An acute perforation closure experiment was conducted using SPF pigs (30 kg body weight). Under general anesthesia, a perforation with an inner diameter of 5 mm was made in the porcine colon using an ESD-dedicated knife. After confirming the collapse of the colon due to deaeration, the collagen sol was immediately delivered to the perforated part via a catheter (similar to [pig stomach perforation closing experiment]). After confirming that the colon was expanded by insufflation, it was rested for 1 hour, and the pig was euthanized, and then the colon was cut and excised. A leak test was performed in which one side of the cut colon was closed with forceps and water was injected from one opening to fill the colon with water.

[Pig stomach ex vivo ulcer protection test]
An ex vivo ulcer protection test was conducted using a porcine excised stomach, which mimics the treatment of ulcers produced by endoscopic treatment with collagen gel. A swab excised stomach of about 60 x 60 mm is submucosally injected with saline using a 23G needle to form a bulge, and a 30 x 30 mm artificial ulcer is created using a scalpel in the center of the excised stomach. Produced. The test piece thus obtained was fixed on an aluminum plate inclined at 60 ° and placed in an incubator (Rcom Max 20; manufactured by Autoelex) set at 37 ° C. and a humidity of 70%. After confirming that the surface temperature of the test piece reached 37 ° C. using an infrared thermometer (IT-545; manufactured by HORIBA, Ltd.), collagen sol ( 3 mL) was delivered. The mixture was allowed to stand for 2 hours to complete the gelation of collagen.
In order to evaluate the adhesion and thickness of the collagen gel formed on the artificial ulcer part, the test piece was subjected to tissue observation. The test piece was fixed with 4% paraformaldehyde aqueous solution and replaced with 20% sucrose aqueous solution, and then embedded with 4% carboxymethylcellulose aqueous solution. This was frozen at −100 ° C. for 5 minutes, and a section having a thickness of 20 μm was prepared using a freezing microtome (CM3050S; manufactured by Leica Microsystems). The obtained sections were air-dried and subjected to hematoxylin-eosin staining. The sections were washed with an aqueous ethanol solution whose concentration was gradually increased from 70% to 99.5%, sealed with a mounting medium (Eukitt; Kindler), and then with an upright microscope (BX53; Olympus). Observed.

(Dynamic viscoelasticity test)
(Viscosity measurement)
The viscosity of the collagen sol was measured by a rotation mode using a dynamic viscoelasticity measuring device (HAAKE MARSIII; manufactured by ThermoFisher Scientific). Collagen sol was filled in a double cone sensor (DC60 / 1Ti, cone angle 1 °) having an inner diameter of 60 mm set at 23 ° C., and rotation at a shear rate of 1 s ⁻¹ was started. The shear rate was gradually increased to 100 s ⁻¹ at a holding time of 20 s for each step, the stress was measured, and the viscosity was calculated from the shear rate and the stress. After confirming that the flow characteristics of the collagen sol were non-Newtonian from the obtained viscosity curve (viscosity vs. shear rate), the viscosity value at a shear rate of 1 s ⁻¹ was adopted.
(Dynamic viscoelasticity measurement)
The gelation rate of the body temperature responsiveness of the collagen sol was measured by dynamic measurement (vibration mode) using a dynamic viscoelasticity measuring apparatus (similar to that used for viscosity measurement). A double cone sensor set at 23 ° C. was filled with collagen sol, and dynamic measurement (frequency, 1 Hz; shear strain, 0.005) with controlled shear strain was started. After 5 minutes, the temperature was increased from 23 ° C. to 37 ° C. over 30 s and kept at 37 ° C. as it was. Changes in storage modulus (G ′) and loss modulus (G ″) were followed throughout the entire process. The time from when the viscoelastic property, which was G ′ <G ″ at room temperature, until the apparatus temperature reached 37 ° C. until G ′ = G ″ was defined as the gel time.

[Penetration test of gel prepared on dish]
The mechanical properties of the collagen gel were evaluated by a mechanical test in which a probe was inserted into the center of the gel. A collagen sol of about 3 g / well (inner diameter 35 mm) was added to a 6-well biological plate, and it was floated on a 37 ° C. water bath and heated. After 30 minutes, the entire plate was covered with parafilm to prevent drying, and left in a 37 ° C. incubator for 24 hours to complete gelation. Using a texture analyzer (TA.XTplus; manufactured by Stable Microsystems), a stainless steel cylindrical probe having a diameter of 5 mm was penetrated into the center of the obtained gel at a speed of 0.2 mm / s to obtain a stress-strain curve. . The elastic modulus was calculated from the slope of the linear region of strain 0.005 to 0.04 in the stress-strain curve.

[Test Example 1]
Using the collagen sol of Example 1, the porcine stomach perforation closing experiment described in the test method was performed. The appearance of porcine stomach perforation, collagen sol delivery, gelation and perforation closure is shown in FIG. It was proved that the perforated part was closed because the stomach was expanded when air was fed immediately after the sol in the perforated part was gelled.
The state of the leaked gastric leak test is shown in FIG. There was no water leakage from the two closed holes, and it was proved that the hole was closed.
FIG. 3 shows an HE-stained image of porcine stomach tissue containing a perforated part. The perforated part was closed so as to be plugged with gel, and it was observed that the gel and the tissue were in close contact.

[Test Example 2]
Using the collagen sols of Examples 1 and 2, the porcine colon perforation closure experiment described in the test method was performed. The appearance of porcine colon perforation, collagen sol delivery, and perforation closure is shown in FIG. It was proved that the perforation was closed because the colon was expanded when air was supplied immediately after the sol in the perforation was gelled.
FIG. 5 shows a state of the leak test of the removed colon. When any sol was used, no water leakage was observed from the two closed perforations, and it was proved that the perforation was kept closed. In addition, when the collagen sol of Example 1 was used for colon perforation closure, the sol sometimes flowed out of the intestine due to the pressure difference generated in the perforated part, and the gel could not be successfully formed in the perforated part. When the collagen sol of Example 2 (1.6%) having a higher concentration than 1.44% was used, gel formation at the perforated part was easier.

[Test Example 3]
Using the collagen sol of Example 3, the porcine stomach perforation closing experiment described in the test method was performed. FIG. 6 shows the state of perforation formation, collagen sol delivery, and perforation closure on the bottom of a porcine gastric ulcer produced by the same method as in Test Example 1. It was proved that the perforated part was closed because the stomach was expanded when air was fed immediately after the sol in the perforated part was gelled. Even when physiological saline was sprayed onto the gel, the gel was not destroyed (FIG. 6).
Further, a perforation with a larger inner diameter (inner diameter 10 mm) was created at another position, and the perforation was closed with a gel (FIG. 6e). The gel was not destroyed when saline was sprayed onto the gel (FIG. 6f). The gel was not broken. It was confirmed that the gel that diffused and adhered to the bottom of the ulcer around the perforation was not peeled off by the injection of physiological saline and protected the ulcer. The collagen sol of Example 3 contained genipin at the same concentration as in Examples 1 and 2, and the strength when gelled was considered to be sufficient for ulcer protection.

[Test Example 4]
Using the collagen sols of Example 1, Example 4, Comparative Example 2 and Comparative Example 3, the ex vivo ulcer protection test for porcine stomach described in the test method was performed. The results are shown in FIG. When the collagen sol of Example 1 and Example 4 was sprayed on the tilted porcine excised gastric ulcer, formation of a collagen gel layer similar to the control test was observed on the submucosal surface. The thickness of the gel layer is 24 ± 4 mm (average value of 10 points ± standard deviation) when the sol of Example 1 is used, and 20 ± 2 mm (average value of 10 points ± when the sol of Example 4 is used. Standard deviation).
In actual surgery, the ulcer part is not always located below. If positioned below, the sol gels without dissipating from the ulcer bottom even if there is time to gel (control in FIG. 7). However, in clinical practice, the ulcer is often located on the side of the stomach. The result of this test example shows that the sol of the example can be protected by a gel layer by rapid gelation even for a lateral ulcer that is easily dissipated by gravity.
On the other hand, when the collagen sol of Comparative Example 2 was used, a portion where the collagen gel layer was not formed was generated, and the thickness of the gel layer was 11 ± 2 mm even in the formed portion (average value of 10 points ± standard). (Deviation). By reducing the NPB concentration, the sodium chloride concentration contained in the sol decreased to 140 mM, the same as PBS, and the body temperature responsiveness of collagen fibrosis decreased (see Test Example 5 and FIG. 8). It was thought that it had dissipated from the ulcer before gelation.
Comparative Example 3 When collagen sol was used, when collagen sol was sprayed onto an inclined swine excised stomach ulcer, collagen sol was dissipated from the ulcer before gelation, and formation of a gel layer could not be confirmed from the tissue section. . It was thought that ulcer protection by gel became difficult due to the decrease in collagen concentration.

[Test Example 5]
A dynamic viscoelasticity test of the collagen sols of Example 1, Example 2, and Comparative Example 2 was performed. The result of evaluating the body temperature responsiveness of gelation is shown in FIG. 8a.
When using the collagen sol of Example 1 and Example 2, G ′ did not change during the first 5 minutes when the apparatus temperature was maintained at 23 ° C., and G ′ increased exponentially immediately after reaching 37 ° C. did.
On the other hand, when the collagen sol of Comparative Example 2 was used, the body temperature responsiveness of collagen fibrosis decreased. This was considered to be because the sodium chloride concentration contained in the sol was lowered to 140 mM, which was the same as PBS, by reducing the NPB concentration.
Next, the dynamic viscoelasticity test of the collagen sols of Example 1, Example 5 and Example 6 (genipin 4 mM, 2 mM and 0 mM, respectively) was performed. FIG. 8 b shows the result of tracking G ′ for 30 minutes from the start of measurement (24.5 minutes after the apparatus temperature reached 37 ° C.). The difference in genipin concentration had little effect on the exponential G ′ increase immediately after reaching 37 ° C., but G ′ gradually increased depending on the genipin concentration after 5 minutes had elapsed after reaching 37 ° C. Increased. On the other hand, in the case where genipin was not added, the increase in G ′ was almost completed 7 minutes after reaching 37 ° C.
From these results shown in FIG. 8, the collagen fibrillation is responsible for the gelation that occurs immediately after the collagen sol contacts the living tissue, and the collagen fiber gel is gradually cross-linked by genipin. It was thought that a strong gel could be obtained.

[Test Example 6]
In order to separately describe the gelation due to collagen fibrillation and the gelation due to genipin crosslinking, the NPB concentration (2 × NPB) of the collagen sol of Example 6 which is 1.44% collagen sol without addition of genipin is set as follows: The dynamic viscoelasticity test was carried out by changing to 4, 1.6 and 1.8 (xNPB), and the gelation behavior due to body temperature responsive fibrosis was examined. The result is shown in FIG. 9a. When 1 × NPB corresponding to PBS, which is widely used as an isotonic solution, was used, no increase in G ′ was observed in about 5 minutes after the apparatus temperature reached 37 ° C., and collagen fibrillation was slow. On the other hand, increasing the NPB concentration accelerated the gelation of body temperature responsiveness depending on the NPB concentration. From the result of plotting the gelation time after reaching 37 ° C. against the NPB concentration (FIG. 9b), it can be seen that the gelation due to collagen fibrillation was accelerated by the increase in the NPB concentration.

[Test Example 7]
Example 1 and Example 6 collagen sols (genipin concentrations of 4 mM and 0 mM, respectively) and the results of penetration tests of gels prepared on dishes using collagen sols having genipin concentrations of 0.2 mM and 1 mM. As shown in FIG. The overall slope of the obtained stress-strain curve increased with genipin concentration, indicating that the strength of the collagen gel was improved by the addition of genipin. As the genipin concentration increased from 1 mM to 4 mM (concentration of Example 1), the increase in slope almost reached its peak (FIG. 10a). In the absence of genipin, the gel was weak and brittle.
On the other hand, the elastic modulus of the collagen gel also increased with the genipin concentration, but showed a monotonic increase to 4 mM (FIG. 10b). The elastic modulus obtained with the sol-derived gel of Example 1 (genipin concentration 4 mM) was about 8 times higher than that of the sol-derived gel of Example 6 containing no genipin.
From the results of FIG. 10, it can be seen that when the genipin concentration is 1 mM, the same gel strength as that of 4 mM of Example 1 is obtained, and an elastic modulus that is 4.0 times higher than that of the gel to which genipin is not added is obtained. Therefore, by adding genipin, a gel harder than the collagen sol to which genipin is not added can be formed in the perforated part.

[Test Example 8]
A sol was prepared by changing the collagen concentration of the collagen sol of Example 6 (collagen concentration: 1.44%) to 0.5, 0.9, 1.2, 1.8, and 2.06%, and dynamically FIG. 11 shows the result of measuring the viscosity at a shear rate of 1 s ⁻¹ by the rotation mode of the viscoelasticity measuring apparatus.
When the collagen concentration was 0.5%, the viscosity was only 0.12 (Pa · s), but when the concentration was increased to 1.44%, the viscosity increased to 6.56 (Pa · s). When the viscosity became 2.06%, the viscosity reached 19.4 (Pa · s). As described above, the viscosity of collagen sol has an exponential change property with respect to the collagen concentration. Therefore, the contradictory properties of collagen sol in the gastrointestinal tract and its ability to be introduced into a capillary tube such as a catheter. It is important to adjust the concentration to meet the above requirements.

[Test Example 9]
Using the collagen sol of Comparative Example 1, a porcine stomach perforation closing experiment was attempted. However, as shown in FIG. 12, the sol discharged from the catheter was already gelled (FIG. 12a), and the collagen gelled in a string was slid down without adhering to the bottom of the ulcer (FIG. 12b). An additional discharge was performed, but a cord-like collagen gel was discharged from the catheter tip (FIGS. 12c and 12d).
The endoscope installed so as to pass through the oral cavity and the esophagus is heated by body temperature, and the catheter inserted therein is also heated to a temperature higher than room temperature. Collagen sols whose body temperature-responsive gelation has been accelerated by the use of high concentrations of NPB and genipin may gel prior to delivery to the affected area, closing biological tissue pores, protecting ulcers and vascular emboli It was considered unsuitable for use.
* Evaluation A. In vivo porcine gastric perforation closure. In vivo porcine colon perforation closure C.I. Ex vivo swine gastric ulcer protection Evaluation of temperature response of gelation Gel penetration test Rotational viscosity measurement

  According to the present invention, three properties suitable for through-hole closure, ulcer physical protection and vascular embolization are (1) long fluidity retention time that can be delivered from ex vivo to in vivo through a catheter, (2) Sharp body temperature responsiveness that gels immediately after delivery, and (3) Hardening after gelation, and the property of fixing to living tissue. Protection and vascular embolization can be performed. The present invention has industrial applicability in the medical field.

Claims (8)

  A biological tissue pore closing sol containing 0.6 mass% to 3 mass% collagen, water, 200 mM to 330 mM sodium chloride and a buffer, and having a pH of 6.0 to 9.0.   An ulcer protective sol containing 0.6% to 3% by mass of collagen, water, 200 mM to 330 mM sodium chloride and a buffer, and having a pH of 6.0 to 9.0.   A vascular embolization sol containing 0.6 mass% to 3 mass% collagen, water, 200 mM to 330 mM sodium chloride and a buffer, and having a pH of 6.0 to 9.0.   The sol as described in any one of Claims 1-3 in which the said buffering agent contains a phosphate.   The sol according to any one of claims 1 to 4, comprising genipin or a genipin derivative in a range of 40 mg / L to 1400 mg / L.   The sol according to any one of claims 1 to 5, wherein the collagen contains telopeptide-removed collagen.   The sol according to any one of claims 1 to 6, comprising 1.4% by mass to 1.7% by mass of collagen and locally administered to a living tissue through a catheter.   The sol according to any one of claims 1 to 7, which gels and adheres to a living tissue when it comes into contact with the living tissue.